Reverse transcriptase activity can be pelleted from non-A, non-B sera by polyethylene glycol 6000 and two high speed centrifugation methods. Particle-associated reverse transcriptase activity was detected in 4 human serum specimens and in plasma-derived products, all of which had been shown to transmit non-A, non-B hepatitis (NANBH) to other humans and/or chimpanzees. Reverse transcriptase activity was also detected in 12 sera from patients with acute or chronic NANBH. In contrast, reverse transcriptase activity was found in only 2 of 49 serum specimens from healthy plasma donors and laboratory workers. Sucrose density grandient fractions of 2 of the infectious human sera (peak reverse transcriptase activity at 1.14 g/ml) transmitted NANBH to chimpanzees. Biochemical and enzymatic data indicate that the NANBH agent is a retrovirus or retrovirus-like agent. A portion of this work has been licensed as an intervention, "Screening Test for Reverse Transcriptase Containing Virus," and a royalty-bearing license has been granted to Electro-Nucleonics, Inc., and E.I. du Pont de Nemours by The Office of Federal Patent Licensing, National Technical Information Service, Department of Commerce. Recently, we have isolated a new mutant carrying a cysE-pyrE linked mutation, designated rfa-2, which results in an increased permeability to hydrophobic agents and a heptose-less lipopolysaccharide structure. Further, we have demonstrated that the rfa-2 mutation is genetically distinct from the rfaD locus and rfa-2 phenotype is not abolished by plasmid carrying the wild type rfaD allele. The rfaD gene product, ADP-L-glycero-D-mannoheptose-6-epimerase, is detectable in crude extracts of rfa-2 mutant strains. An automated sugar analyzer has been developed in our laboratory which allows us to follow directly the conversion of sugar intermediates involved in the biosynthesis of L-glycero-D-mannoheptose. The cloned rfaD gene has been expressed in several expression systems, allowing further characterization of the rfaD gene product.